Project Summary Otitis media (OM) affects more than 90% of children by the age of five and is the most common indication for antimicrobial therapy in children. OM is also the most common cause of hearing loss in children and can lead to significant educational and speech delays if left untreated. Surgical interventions for OM can cause significant morbidity including pain, facial paralysis, meningitis, and brain abscess formation. With an annual cost of $5 billion, OM has a large impact on health care nation-wide. Although the impact of OM is significant, the cellular and molecular events that regulate this disease process are still poorly understood. Investigations have been limited by having only a singular tool to conduct in vitro cell culture experiments. The objective of this R21 study is to develop, characterize, and compare immortalized middle ear (ME) epithelial cell lines derived from normal tissue obtained from both adult and pediatric cochlear implant patients without a history of OM and from patients with recurrent OM (ROM) and OM with effusion (OME). These cell lines will then be made available to the OM research community for further comparative analyses. Recent in vitro cell line, in vivo animal, and clinical data from a cohort study of OM patients and control CI subjects strongly support a key role for MUC5B and miRNA-146 in the pathophysiology of OM. The effect of recombinant TNF-? and IL-1? on MUC5B and miRNA146 expression levels, as they relate to the pathogenesis of OM, will be measured in these novel cell lines. These new tools will enhance, and potentially even change, assumptions which have been based on a narrow window of investigation. The cell lines established by this project will aid the OM research community to understand the differences between adult and pediatric patients and uncover potential mechanisms differentiating patients who suffer from OM from those who do not.